OPEN ACCESS International Journal of Pharmacology

ISSN 1811-7775 DOI: 10.3923/ijp.2017.1086.1091

Research Article Antimicrobial and Antioxidant Efficacy of Hochst. ()

1Wilfred Otang-Mbeng and 2Anthony Jide Afolayan

1School of Biology and Environmental Sciences, Faculty of Natural Sciences and Agriculture, University of Mpumalanga, Mbombela Campus, P/bag X11283, 1200 Nelspruit, Mpumalanga, South Africa 2Medicinal and Economic Development (MPED) Research Centre, Department of Botany, University of Fort Hare, Private Bag X1314, 5700 Alice, South Africa

Abstract Background and Objective: Acokanthera oblongifolia is an evergreen medicinal shrub used for snakebites, itches, wounds and internal worms and the relief of itchy conditions and other skin disorders by the Mpondo and Xhosa tribes in South Africa. The objective of this study was to investigate the efficacy of the extracts against selected pathogens that cause human skin disorders and evaluation of the antioxidant capability for validation of folk uses of the plant. Materials and Methods: The agar diffusion and micro-dilution methods were used to determine the antimicrobial activities of the extracts against selected bacteria and fungi. The data were subjected to one way analysis of variance (ANOVA) and Duncan’s Multiple Range Test (DMRT) was used to determine significant differences (p<0.05) among treatment means. Results: The highest antibacterial activity (inhibition zone diameter >19 mm) was obtained with the acetone extract against Pseudomonas aeruginosa, Shigella sonnei, Shigella flexneri, Bacillus cereus, Streptococcus pyogens and Bacillus subtilis; by the ethanol extract against B. cereus. None of the extracts was active against the tested fungi, apart from the acetone extract which showed strong inhibitory activity against Candida glabrata. The ethanol extract showed a higher ABTS scavenging than those of gallic acid and BHT at concentrations lower than 0.15 mg mLG1. Conclusion: The in vitro antibacterial activity of the acetone extract of A. oblongifolia against the tested pathogens has provide scientific evidence to justify the ethnomedicinal use of A. oblongofolia against skin disorders in the study area and also indicate that the plant is a potential source for the development of antimicrobial and antioxidant agents.

Key words: Acokanthera oblongifolia, skin ailments, ethnomedicinal investigation, free-radical scavenging activity, antimicrobial activity

Received: April 07, 2017 Accepted: June 06, 2017 Published: October 15, 2017

Citation: Wilfred Otang-Mbeng and Anthony Jide Afolayan, 2017. Antimicrobial and antioxidant efficacy of Acokanthera oblongifolia Hochst. (Apocynaceae). Int. J. Pharmacol., 13: 1086-1091.

Corresponding Author: Wilfred Otang-Mbeng, School of Biology and Environmental Sciences, Faculty of Natural Sciences and Agriculture, University of Mpumalanga, Mbombela Campus, P/bag X11283, 1200 Nelspruit, Mpumalanga, South Africa Tel: +27 (0) 130020235

Copyright: © 2017 Wilfred Otang-Mbeng and Anthony Jide Afolayan. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files. Int. J. Pharmacol., 13 (8): 1086-1091, 2017

INTRODUCTION MATERIALS AND METHODS

Medicinal plants have been used by mankind for the This study was carried out from December 2015 to management of diverse diseases since antiquity1. According February 2017 in the Amathole District, Eastern Cape, to the World Health Organisation, about 20,000 plant species South Africa. are used as medicines1,2. The widespread usage of medicinal plants in many cultures of the world could be attributed to a Extraction procedure: The leaves of A. oblogifolia were number of reasons including affordability and limited sterilized with 70% analytical grade ethanol and then washed availability of Western medicine as well as the popular believe with distilled water. Dried, pulverised leaf samples were that herbal medicines are less toxic since they are natural1. shaken for 24 h in ethanol and acetone within separate Natural products, whether standardised plant extracts or pure conical flasks placed on an orbital shaker. The solutions were compounds provide opportunities for the development of filtered through Whatman No. 1 filter paper in a Buchner new drug leads partly due to their unmatched chemical funnel and the filtrates were dried in a rotavapor at 40EC 1 diversity3,4. Natural products and related drugs are used to under reduced pressure. A 20 mg mLG stock solution was treat 87% of infections caused by bacteria, immunological formed by dissolving the extracts in their respective solvents disorders and cancer cases and about 25% of prescribed drugs of extraction. in the world originate from medicinal plants5,6. Microorganisms: Antibacterial studies were conducted Acokanthera oblongifolia Hochst. (Apocynaceae) is a using five Gram-positive bacteria (Staphylococcus aureus, common medicinal plant used for the treatment of various Streptococcus pyogenes, Enterococcus faecalis, Bacillus cereus diseases in the Eastern Cape Province of South Africa. It is an and Bacillus subtilis) and six Gram-negative bacteria ornamental shrub with densely clustered white flowers (Salmonella typhimurium, Escherichia coli, Pseudomonas located in leaf axils. The plant can grow to a height of 3 m as aeruginosa, Shigella sonnei, Shigella flexneri and Klebsiella a shrub and up to 6 m as a small tree. The dark-green leaves pneumonia). Antifungal activities were evaluated using are elliptic, opposite and leathery in texture. Because all parts Candida krusei and Candida glabrata. The choice of these of the plant are toxic, it has been used as arrow poison and microbes was attributed to their significance as pathogens related species in other parts of the world have been reported that cause human skin disorders. The microbes were American as causing deaths7. The ripe berries may cause salivation, Type Culture Collection (ATCC) purchased from Total vomiting and abdominal pain when ingested. The milky the Laboratory, South Africa. sap is irritant to the skin and eyes8. Hence the plant is also known as poison bush, bushman’s poison, or poison tree Antimicrobial susceptibility assays: Nutrient agar and because of its toxicity7. The leaf or wood decoction of the plant Sabouraud Dextrose Agar (SDA) media were prepared as per is used in local medicinal treatments for snakebites, itches, the recommendations of the manufacturer and poured into wounds and internal worms and an ointment made from sterilized disposable petri dishes under aseptic conditions. The the root scrapings of Acokanthera oblongifolia is used for labeled plates were inoculated with 100 µL of 0.5 Mcfarland the relief of itchy conditions and other skin disorders by the solutions of the respective organisms and loaded with extracts Mpondo and Xhosa tribes of the Eastern Cape Province of of A. oblongifolia (50 mg mLG1) into 6 mm wells. Positive 8 South Africa . In view of the importance of A. oblongifolia in controls for fungi and bacteria were nystatin and gentamycin ethnopharmacology and the dearth of scientific literature of respectively. The plates were incubated at 37ºC and the zones the in vitro medicinal potential of A. oblongifolia, the present of inhibition were recorded after 24 h. The Minimum Inhibitory study was undertaken to investigate the efficacy of the Concentration (MIC) of the plant extracts was determined acetone and ethanol extracts of the plant against selected using 96 well microtitre plates9. The least concentration of pathogens that cause human skin disorders and evaluation of plant extract that killed the microbes was considered as MIC. the antioxidant capability for validation of folk uses of the plant. This study has provided new evidence necessary for Assay of DPPH scavenging activity: The DPPH the scientific validation of the claimed medicinal potential of radical-scavenging activity of the test extracts was examined A. obolongifolia extracts and a basic understanding of the as previously described by Erukainure et al.10. Different extract plant‘s efficacy which may lend further support to the concentrations (0.025-0.5 :g mLG1) were added to equal widespread use of traditional medicine in health care systems volumes of methanolic solution of DPPH (100 :M) and in South Africa. the mixture was left for 30 min under darkness at room

1087 Int. J. Pharmacol., 13 (8): 1086-1091, 2017 temperature. Vitamin C and Rutin were used as standard Absorbance of the control- Absorbance of the sample (2) controls. Three replicates were made for each test sample. The ABTS+ scavenging activity (%) = 100 Absorbance (A) was measured after 30 min at 518 nm and the Absorbance of the control percentage antioxidant activity was calculated according to the Eq. 1: Reducing power: Different extracts concentrations (0.025-0.05 :g mLG1) prepared in distilled water were mixed Absorbance of the control- with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL Absorbance of extract or standard (1) 4 DPPH scavenging activity (%) = 100 potassium ferricyanide (1% w/v) . The resulting mixture was Absorbance of the control incubated at 50EC for 20 min, followed by the addition of 2.5 mL of trichloroacetic acid (10% w/v). This was then The IC values denote the concentration of sample which 50 centrifuged at 3000 rpm for 10 min and the supernatant is required to scavenge 50% of DPPH free radicals. The IC 50 (2.5 mL) was mixed with an equal volume of distilled water values were calculated by linear regression of plots, where the and 0.5 mL of FeCl3 (0.1% w/v) and the absorbance (at 700 nm) abscissa represented the concentration of the tested plant was read. Vitamin C and gallic acid were used as positive extracts and the ordinate the average percent of scavenging controls. capacity from three replicates.

Statistical analysis: All tests were done in triplicates and the ABTS scavenging activity: For the ABTS assay, an aqueous zones of inhibitions were subjected to one way analysis of solution of ABTS (7 mmol LG1) was made and ABTS+ was variance (ANOVA) and Duncan’s Multiple Range Test (DMRT) produced by reacting the ABTS stock solution with was used to determine significant differences among 2.45 mmol LG1 potassium persulfate (final concentration) and treatment means. Minitab program (version 12 for windows) allowing the mixture to stand in the dark at room temperature was the statiscal software that was used. p-values<0.05 were for 12-16 h before use11. The ABTS+ stock solution was diluted 3 with 80% methanol to an absorbance of 0.70±0.02 at 734 nm. regarded as significant . After the addition of 4.85 mL of diluted ABTS+ to 0.15 mL of 1 RESULTS 10-fold diluted samples (final concentration 0.025-0.5 :g mLG of each extract were added, at an equal volume dry material), the absorbance reading was taken 6 min after the initial Antimicrobial assays: The inhibition diameters and the MICs mixing. Gallic acid and BHT were used as positive controls. The of the extracts and positive controls are shown in Table 1. The ABTS activities of the samples were calculated according to acetone extract of A. oblongifolia showed the most potent the Eq. 211: antibacterial activity (inhibition zone diameter >19 mm)

Table 1: Inhibition zone diameters and MICs of A. oblongifolia extracts against the test bacteria and fungi Ethanol Acetone Positive control ------ZI MIC ZI MIC ZI MIC Microorganisms (mm) (mg mLG1) (mm) (mg mLG1) (mm) (mg mLG1) Gram negative bacteria Salmonella typhimurium 12±1.1 5.00 17±1.7 2.50 25±1.0 0.01 Escherichia coli 17±2.1 2.50 16±1.1 2.50 25±0.7 <0.01 Pseudomonas aeruginosa 15±1.1 2.50 20±2.0 0.01 24±2.1 0.01 Shigella sonnei 15±1.1 2.50 20±1.0 0.01 24±2.6 <0.01 Shigella flexneri 12±1.0 5.00 20±1.3 0.01 24±1.1 <0.02 Gram positive bacteria Enterococcus faecalis 15±1.1 5.00 23±1.7* 0.01 24±1.4 <0.10 Bacillus cereus 20±1.1 0.01 20±1.4 0.01 24±1.6 <0.01 Streptococcus pyogens 15±2.0 5.00 20±1.2 0.01 23±2.1 <0.01 Bacillus subtilis 15±1.2 5.00 20±2.0 0.01 22±1.2 0.02 Klebsiella pneumoniae 15±1.1 5.00 15±1.5 5.00 24±2.2 <0.01 Staphylococcus aureus 15±1.3 5.00 15±2.0 5.00 24±0.6 <0.10 Fungal strains Candida krusei N.A. N.A. N.A. N.A. 21±1.2 0.02 Candida glabrata N.A. N.A. 20±1.3 0.02 20±1.7 0.02 Values are Mean±SD of triplicates experiments, N.A.: Not active, *Not significantly different form positive control (p<0.05), ZI: Zone of inhibition, MIC: Minimum inhibitory concentration

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120 Ethanol Rutin 1.8 Ethanol Gallic acid Acetone Vitamin C Acetone Vitamin C 1.6 100 1.4 80 1.2 1.0 60 0.8 40 0.6 Percentage of inhibition Percentage Percentage of inhibition of Percentage 0.4 20 0.2 0 0.0 0.025 0.05 0.1 0.2 0.5 0.025 0.05 0.1 0.2 0.5 Concentration (mg mLG1 ) Concentration (mg mLG1 )

Fig. 1: DPPH scavenging activity of A. oblongifolia extracts Fig. 3: Reducing power of A. oblongifolia extract

Ethanol Gallic acid Results of antioxidant assays: The concentration required to 100 Acetone BHT attain 50% radical-scavenging effect (IC50) was determined 80 from the results of a series of concentrations tested and are 60 presented in Table 2. A lower IC50 value corresponds to a larger 40 scavenging activity. 20 0 DPPH radical-scavenging activity: Scavenging activity was 0.025 0.05 0.1 0.2 0.5

Percentage of inhibition Percentage -20 expressed as percentage of inhibition of DPPH free radical 1 -40 Concentration (mg mLG ) (Fig. 1). Higher % inhibition indicates better scavenging

-60 activity or antioxidant potential. The results of the DPPH assay also showed that the scavenging activity of the Fig. 2: ABTS scavenging activity of A. oblongifolia extracts acetone extract was higher than that of the ethanol extract and was not significantly different from those of vitamin C Table 2: Scavenging activity (mg mLG1) of the ethanol and acetone extracts of A. oblongifolia and rutin. Reducing power DPPH ABTS Parameters (mg mLG1) (mg mLG1) (mg mLG1) ABTS scavenging activity: The ABTS scavenging activity of Ethanol 0.16 0.15 0.12 the ethanol extract of Acetone 0.18 0.12 0.15 A. oblongifolia was higher than those Vitamin C 0.19 0.13 ND of the acetone extract, BHT and Gallic acid, except at BHT ND ND 0.11 concentrations greater than 0.05 mg mLG1 (Fig. 2). The Gallic acid 0.14 ND 0.14 least DPPH scavenging activity was exhibited by the Rutin ND 0.13 ND ND: Not determined acetone extract which decreased in a dose-dependent manner. against Bacillus cereus, Pseudomonas aeruginosa, Shigella flexneri, Shigella sonnei, Streptococcus pyogens and Reducing power assay: The reducing power of both Bacillus subtilis. The acetone extract strongly inhibited the ethanol and acetone extracts of A. oblongifolia Candida glabrata but was not active against Candida krusei. increased with increasing concentration up till a The ethanol extract was active against B. cereus with an concentration of 0.2 mg mLG1, after which there was a inhibition diameter of 20±1.1 mm but was not active against sharp fall in reducing power at concentrations greater any of the tested fungi. than 0.2 mg mLG1 (Fig. 3). The reducing power of the The least antibacterial activity was exerted by the ethanol ethanol extract was higher than that of vitamin C within extract against Salmonella typhimurium and Shigella flexneri the range of 0.025-0.25 mg mLG1, while the least with zones of inhibition of 12±1.1 and 12±1.0 mm, reducing power was exhibited by the acetone respectively. extract.

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DISCUSSION or over-treated with antibiotics leading to great disability19. These limitations have fostered the search of better In the current study, the acetone extract of A. oblongifolia antimicrobials and much attention is now being directed proved to be active against Pseudomonas aeruginosa, towards medicinal plants. Shigella sonnei, Shigella flexneri, Bacillus cereus, The human skin is exposed to many environmental Streptococcus pyogens, Bacillus subtilis and Candida glabrata. pro-oxidants including solar radiation, pollutant chemicals, Although the antimicrobial activity of A. oblongifolia extracts drugs, food additives and cosmetic products, which generate has not been reported in previous scientific literature, studies Reactive Oxygen Species (ROS). Natural skin antioxidant by various authors have documented the in vitro medicinal molecules such as vitamin E, glutathione, catalase, vitamin C, potential of other members of the Acokanthera. etc. can neutralise the deleterious effects generated by Chaurasia and Sharma12 reported that the methanol, acetone reactive oxygen species. However, when this homeostatic and chloroform extracts of Acokanthera oppositifolia showed mechanism is overwhelmed, the resultant increase in reactive significant antibacterial activity (p = 0.02) against two human oxygen species may cause inflammatory skin diseases20. pathogenic bacteria: E. coli (gram negative) and B. subtilis Although synthetic antioxidants such as butylhydroxyanisole (gram positive) by disk diffusion assay. The range of and butylhydroxytoluene are used to treat ROS-mediated antibacterial activity varied from 17±0.73-25±0.8 mm in disorders, the health risks associated to synthetic antioxidants E. coli and 18±0.16-24±0.14 mm zone of inhibition in has motivated the search for safer antioxidants from plants. B. subtilis. In this study, ABTS scavenging activities of the acetone The antimicrobial activity of different plant extracts and ethanol extracts were comparable to the standard against pathogens that cause skin disorders in humans controls. Adedapo et al.21 reported the presence of phenolics have previously been reported by other authors. Nesy and in A. oppositifolia and that the ABTS activity of the methanol Mathews13 assessed the antimicrobial and antioxidant extract was not significantly different to that of BHT. These activity of Thevetia neriifolia flowers against seven studies taken together suggest that the antioxidant activity pathogenic microbes implicated in human skin diseases. A A. oblongifolia could be attributed to the presence of poor antimicrobial activity was observed at lower doses phenolics. The antioxidant activity of polyphenols is mainly (1.25-2.5 mg per well), but at medium concentration due to their redox properties which neutralise free radicals, (5 mg per well), S. aureus was significantly inhibited (p = 0.03) quench singlet and triplet oxygen and decompose in a manner comparable to standard Gentamicin. The peroxides21. methanolic extract of Tabernaemontana heyneana showed The antibiotic and free-radical scavenging effects of limited in vitro antibacterial activity against Staphylococcus A. oblongifolia extracts as revealed in this study implies that aureus and Proteus vulgaris14. The polyphenol extract of the the plant is a potential source of leads compounds that may edible flower of Sesbania grandiflora reportedly inhibited be used for the development of alternative remedies for the Shigella, exneri, S. aureus and E. coli; S. aureus was the most treatment of skin ailments. However, taking into consideration susceptible with a minimum inhibitory concentration of the reported irritancy of the milky sap of A. oblongifolia to the 0.013 mg mLG1 15. The ethanol extract of the flower of skin and eyes8, the dosage and mode of application of the sap Catharanthus roseus was reported to possess wound should be treated with caution. Hence, In vitro toxicity studies healing activity in Sprague Dowley rats16. The antimicrobial of A. oblongifolia extracts are highly recommended. activity of plant extracts against microbes implicated in human skin disorders were attributed to the presence of CONCLUSION polyphenolics like kaemferol, quercetn, kaempferol and quercetin 7-o-galactoside17. In this study, the acetone extract of A. oblongifolia Infectious diseases of the skin and mucous membranes showed the most potent antibacterial activity against are common in areas characterised by lack of potable water, Bacillus cereus, Pseudomonas aeruginosa, Shigella flexneri, poor sanitation and little hygienic food habits. Many health Shigella sonnei, Streptococcus pyogens and Bacillus subtilis. centers in the Eastern Cape Province of South Africa are often The acetone extract strongly inhibited Candida glabrata but run by nurses who have very limited training in diagnosing was not active against Candida krusei. The ethanol extract dermatologic conditions18. This has led to an uproar in the was active against B. cereus but was not active against any of prevalence of skin diseases such as dermatitis, scabies, acne, the tested fungi. The DPPH scavenging activity of the acetone urticaria and prurigo. These diseases are either under-treated extract was similar to those Rutin and Vitamin C. The ABTS

1090 Int. J. Pharmacol., 13 (8): 1086-1091, 2017 scavenging activity of the ethanol extract of A. oblongifolia 9. Otang, W.M., D.S. Grierson and R.N. Ndip, 2012. Antifungal was higher than those of the acetone extract, BHT and gallic activity of Arctotis arctotoides (L.f.) O. Hoffm. and Gasteria acid, except at concentrations greater than 0.15 mg mLG1. bicolor Haw. against opportunistic fungi associated with human immunodeficiency virus/acquired immunodeficiency SIGNIFICANCE STATEMENT syndrome. Pharmacogn. Mag., 8: 135-140. 10. Erukainure, O.L., O.V. Oke, A.J. Ajiboye and O.Y. Okafor, 2011. Nutritional qualities and phytochemical constituents of This study discovers the antibiotic and free-radical Clerodendrum volubile, a tropical non-conventional scavenging effects of A. oblongifolia extracts that can be vegetable. Int. Food Res. J., 18: 1393-1399. beneficial in the search of safer, effective and alternative 11. Wintola, O.A. and A.J. Afolayan, 2011. 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